Conservation of knotted and slipknotted topology in transmembrane transporters

Abstract

The existence of nontrivial topology is well accepted in globular proteins but not in membrane proteins. Our comprehensive topological analysis of the Protein Data Bank structures reveals 18 families of transmembrane proteins with nontrivial topology, showing that they constitute a significant number of membrane proteins. Moreover, we found that they comprise one of the largest groups of secondary active transporters. We classified them based on their knotted fingerprint into four groups: three slipknotted and one knotted. Unexpectedly, we found that the same protein can possess two distinct slipknot motifs that correspond to its outward- and inward-open conformational state. Based on the analysis of structures and knotted fingerprints, we show that slipknot topology is directly involved in the conformational transition and substrate transfer. Therefore, entanglement can be used to classify proteins and to find their structure-function relationship. Furthermore, based on the topological analysis of the transmembrane protein structures predicted by AlphaFold, we identified new potentially slipknotted protein families.

Figure 1

Matrix presentation of protein knotting fingerprint. Each entry in the matrix indicates the knot type formed by one continuous subchain shown by green color (or by orange for 4₁ knot in, e.g., Figs. 5 and 6). In each case, the subchain starts with the N-terminal amino acid at position x and ends with the C-terminal amino acid at position y, indicated on the horizontal and vertical axes, respectively. Equivalently, this subchain can be interpreted as a part of the diagonal, delimited by the corresponding coordinates x and y, where the entire diagonal corresponds to the entire protein backbone. Left, an example of knotted (a 3₁ knot) protein. The green entry in the lower left-hand corner, which corresponds to the entire protein, indicates that the corresponding chain or subchain is knotted. The knot core is defined as the shortest subchain that still forms a knot. The two remaining parts of the chain form knot tails. Right, an example of slipknotted protein. Notice that in the case of slipknots, the entire protein is unknotted (the element in the lower corner is not colored), but as one terminal is trimmed to some extent, the remaining fragment forms a trefoil knot, denoted S3₁. The corresponding subchain entries are therefore colored in the matrix. Above diagonal, schematic drawings of trefoil knot and slipknot illustrate which parts of the chains constitute the knot cores (blue line), the knot and slipknot tails (black line), and the slipknot loop (orange line). More details about the fingerprint can be found in (8). To see this figure in color, go online.

Figure 2

Classification and entanglement (type of topology) of membrane proteins based on OPM database. Proteins with nontrivial topology are found only within α-helical polytopic class of transmembrane proteins; they are part of four superfamilies (out of 140). Details about topology conservation in each of the entangled superfamilies are in Table 1. To see this figure in color, go online.

Figure 3

Analysis of all secondary active transporters in mammals and humans (SLC tables) shows that approximately 25% of all SLC proteins possess nontrivial topology. The data are grouped according to clans and families from the Pfam database. Additional details are provided for the APC superfamily (CL0062 by Pfam nomenclature). Specifically, NCS2-like proteins are marked with a darker color (the rest of the clan has the LeuT fold). All families without a clan are grouped together (no clan label). If there is a protein with resolved structure in the family, the topology (knotted/unknotted) is given. If a structure is not available, no information about the topology is given (no info label). To see this figure in color, go online.

Figure 4

Knotted protein backbone in sodium/calcium exchangers (NCX; PF01699). (A) The matrix presentation of a protein knotting based on PDB: 4KPP (from Archaeoglobus fulgidus), a 3₁ knot (KC, knot core (blue); KT, knot tail (black)). The color scale shows the dominant knot type formed by a given subchain and the frequency (shown via the color opacity, the green and orange colors are used for knots 3₁ and 4₁, respectively) of its formation. (B) Side view of the knotted protein from Archaeoglobus fulgidus (PDB: 4KPP). The protein knot core is colored blue and knot tails in gray. (C) Top view of the same protein. Only knotted core (TM1–TM8) is shown. (D) Schematic presentation of the knotted protein. The binding site is shown in the middle with a black square. Conserved glutamic acid and proline residues are shown as red circles. (E) Cation binding site (inward-open structure (PDB: 4KPP from Archaeoglobus fulgidus) without the cation bound; outward-open structure (PDB: 5HWY from Methanococcus jannaschii) with cation bound). Conserved glutamic acid and proline residues are shown in a stick representation. (F) Position of the knot core in different structures of the same protein (UniProtKB: Q57556; based on nine PDB structures). To see this figure in color, go online.

Figure 5

Conformation dynamics in LeuT superfamily with S3₁4₁3₁ slipknot motif based on outward (PDB: 3TT1) and inward-open state (PDB: 3TT3). (A) Matrix and (B) structure with denoted 3₁ slipknot territory determined based on the inward-open state (PDB: 3TT3). ST, slipknot tail (green, TM1a); SL, slipknot loop (orange, TM1b); KC, knot core (blue); KT, knot tail (gray/black) with their amino acid ranges written in square brackets. The schematic representation of the slipknot is shown above diagonal. (C) superimposed TM1ab of inward-open and outward-open structures. G24 is the residue of the hinge region and the last residue of the 3₁ slipknot loop in the outward-open state. L29 is the last residue of the 3₁ slipknot loop in an inward-open state. In outward-open conformation, the slipknot tail closes the internal gate and blocks the substrate. In an inward-open state, it moves up and allows the substrate to leave. Short alignment compares residues of slipknot 3₁ in inward- to outward-open states. On the right, there is a schematic representation of the slipknot 3₁ (with marked positions of the key residues, G24 and L29, and the substrate) in inward (upper) and outward (lower) conformations. (D) Matrix and (E) structure of inward-open structure (PDB: 3TT3) with denoted position of 4₁ slipknot. (F) (Upper panel) View of the structures from the extracellular side: in an inward-open state, 4₁ slipknot loop closes the outside gate; in an outward-open state, it moves aside and opens the gate. (Lower panel) Schematic representation of the slipknot 4₁ and position of its loop in inward- (left) and outward-open (right) states. The dashed circle shows the location of the outward gate. (G) Location of the second slipknot 3₁ on the matrix. Note that it almost coincides with the first slipknot 3₁; however, its knotted core is longer. Therefore, the second slipknot 3₁ is found on a different protein subchain. (H) Schematic presentation of the position of slipknot loop 4₁ and slipknot tail 3₁ in inward-open and outward-open structures. Both slipknots 4₁ and 3₁ are shown simultaneously. To see this figure in color, go online.

Figure 6

Potentially different slipknot motifs (3₁3₁ and 3₁4₁3₁) are detected in uracil transporters (inward-open state; PDB: 3QE7). (A) Matrix presentation of protein topology with S3₁3₁ motif marked. (B) Structure colored according to S3₁3₁ motif. (C) Top view of the inward-open structure with colored S3₁3₁ motif. The dotted line shows the border between core and gate domains. Uracil molecule located at the binding site is shown by stick presentation. (D) Matrix presentation of protein with marked 4₁ slipknot territories (3₁4₁3₁ motif) located at the threshold border. ST, slipknot tail (green); SL, slipknot loop (orange); KC, knot core (blue); KT, knot tail (gray/black) with their amino acid ranges written in square brackets. The schematic representation of each slipknot is given in the upper part of the matrix. (E) Structure colored according to 4₁ slipknot. (F) Magnified view of the binding site. Uracil molecule is shown with thick sticks. Residues within 4 Å from the uracil are shown as sticks. The structure is colored according to the slipknot S3₁3₁ motif. To see this figure in color, go online.

Figure 7

Example of different slipknot motifs in SDF transporters, based on inward-open (PDB: 4P19) and outward-open (PDB: 4IZM) state. Matrix presentation of protein in inward-open state (A) and in outward-open (C) shows respectively one and two slipknot territories, motif S3₁ (A) and S3₁3₁ motif (C). The schematic representation of each slipknot in the upper part. ST, slipknot tail (green); SL, slipknot loop (orange); KC, knot core (blue); KT, knot tail (gray/black). The number of residues is written in square brackets. (B) illustrates the difference in protein structures for inward-open state (one slipknot) and outward-open state (two slipknots). Structures are colored according to the slipknot topology. (D) Magnified view of the binding site in inward and outward states. (E) Schematic drawing of the 3₁ and 3₁3₁ slipknots. To see this figure in color, go online.

Figure 8

Inward- and outward-open states in 2HCT can be detected by different range of 3₁ slipknot territory. (A and B) Matrix presentation of protein topology (PDB: 5A1S) shows deep and shallow 3₁ slipknots, corresponding to inward- and outward-open states, respectively (notice the difference in the orange loop and its representation on the diagonal). ST, slipknot tail (green); SL, slipknot loop (orange); KC, knot core (blue); KT, knot tail (gray/black). The schematic representation of each slipknot is given in the upper part of the matrix. (C) Protein structures (colored according to the slipknot topology) showing that in inward-open state, slipknot loop is longer and exits to the extracellular side together with the substrate; in outward-open state, slipknot loop is shorter and moves up toward the extracellular side. (D) View from the intracellular side. (E) Schematic drawing of slipknot geometry in inward- and outward-open states. To see this figure in color, go online.